Sustained phenotypic correction of genetic defects requires a safe means of gene replacement. To date, many of these gene correction strategies use integrating lentiviruses or retroviruses for long-term gene replacement, although their clinical applications remain limited because of potential for viral-associated oncogenesis.
Gene correction strategies have attempted to use hybrid Adenovirus/Adeno-associated viruses (Ad/AAV) to combine the capacity, tropism and ease of production of adenovirus (Ad) with adeno-associated virus's (AAV's) ability for site-specific integration (SSI) into chromosome 19 AAVS1. Although the AAV Rep78 protein is required for SSI, the AAV Rep78 protein has the disadvantage of an inhibitory effect on Ad replication, particularly when co-expressed within the Ad backbone. This has lead to difficulty in the prior art in generating an integrating transgene within the back-bone of a single hybrid virus, such as Ad/AAV.
While an Adenovirus carrying the AAV cis acting elements can be constructed, construction of an Adenovirus carrying the Rep expression cassette has met with only limited success. Work by various authors has shown that coinfection with AAV in general, and Rep protein expression in particular, results in a 10% to 40% decrease in Adenoviral replication. Further, during co-infection of Ad and AAV, Rep protein has been shown to co-localize to Adenoviral replication centers and prevent their maturation. As a result, strategies to construct an Ad/AAV carrying Rep have focused on controlling Rep expression. The few successes reported have utilized tightly regulated expression systems, within a helper dependent Adenoviral vector. Although these vectors are free of adenoviral genes, they however need a helper virus for replication. Also, construction of a first generation Adenovirus carrying Rep has proved to be more difficult. Several reports exist of unsuccessful strategies for the construction of a first generation Ad carrying Rep.
In particular, a stable first generation adenovirus carrying AAV Rep78 has so far not been reproducibly constructed. Most viruses either fail to grow, showing no signs of viral replication (Ueno et al. (2000) Biochemical and Biophysical Research Communications 273(2):473-478), grow slowly, or are unstable, acquiring deletions within the Rep gene (Zolotukhin (2005) Human Gene Therapy 16(5):551-557). In one report, analysis of two clones bearing deletions revealed no overlap of the deletion sites within the Rep ORF (Zhang et al. (2001) Gene Ther. 8:704).
Thus there remains a need for new compositions and methods for safe, site-specific gene integration for applications that include gene therapy, vaccine, etc.